Phosphors typically comprise one or more rare earth metals in a host material. Up-converter phosphors emit light in the visible wavelength radiation range (550-800 nanometers) when excited by long wavelength radiation, e.g., light in the infrared wavelength spectrum. This is accomplished by multiple absorption of infrared photons and energy transfer between the absorbing and emitting ions. For example, it is known that yttrium fluorides doped with certain activator couples such as ytterbium and erbium, will be excited by 0.98 micron wavelength radiation. Such radiation can be provided by semiconductor lasers.
Such phosphors have been tried as phosphorescent labels for biological assays. Detection methods for macromolecules such as proteins, drugs, polynucleotides and the like include an analytical reagent that binds to a specific target macromolecular species and produces a detectable signal, which is provided by a label such as a radioisotope or a covalently-linked fluorescent dye or phosphor.
Up-converting phosphors have several advantages over other known materials, such as radioisotopes and covalently-linked fluorescent dyes, for such label applications. Radioimmunoassays, while they are sensitive, use radioactive materials which are potential health hazards for the operators of the tests, and they also require special handling and present expensive disposal problems. Radioisotopes are unstable, and they do not produce strong signals in the ultraviolet, infrared or visible portions of the electromagnetic spectrum, and thus they cannot be used for methods including microscopy, image spectroscopy and flow cytometry that employ optical methods for detection of the label.
Fluorescent labels have come into widespread use for such assay methods. They include small organic dye molecules that can be illuminated with light of a particular excitation frequency, so that they give off emissions that can be detected by electro-optical sensors. However, these dyes have a short lifetime and they bleach in the presence of light.
The use of up-converting phosphors for immunoassays has been disclosed. These phosphors can be excited by photons of a frequency which can be provided by inexpensive near-infrared laser diodes or light-emitting diodes for example, and they emit light of a lower frequency band, in the visible range. Thus the photons of the emitted radiation are of higher energy than the excitation energy, and the emitted radiation is "up-shifted" from the excitation radiation. This reduces the background noise of the visible emission signal. Solid state diode lasers can be tailored to operate at a variety of wavelengths in the near infrared range, and they are inexpensive.
In order to use them as labels, the phosphor particles should be small, such as one micron or less, and they should desirably have a uniform particle size and morphology. The size, weight and morphology of the particles are important because they affect the strength of particle binding, and the specificity of the separation process of the assay. Further, since in an assay application each of the particles should have a like number of active binding sites, it is also desirable that the particles be of similar size.
Small particle size oxide and oxysulfide up-converter phosphors are known that have uniform particle size of one micron or less, and the particles are all spherical.
Fluoride up-converter phosphors are more efficient than other phosphor hosts, such as the oxides and oxysulfides, and they do not saturate as readily at high infrared flux. Since newer laser diodes have high power, saturation of the phosphor probes is becoming a limitation in the sensitivity of assays. However, such fluoride up-converter phosphors have not been available up to the present time. Attempts to prepare fluoride phosphors by reaction with ammonium fluoride have not been successful.
Submicron sized phosphor particles are also useful as pigments for ink jet printer inks. The phosphor particles can be coated or encapsulated so that they can be suspended in an ink formulation. However, the present phosphors are only available in particles that are about 10 microns in size. Since ink formulations require the phosphor particles to be about 1 micron or less in size, the particles must be milled to reduce their size. Thus at present, small particle size, e.g., less than one micron size particles, fluoride phosphors are only available as milled particles. Milling does not produce uniform particle sizes however, but rather a gaussian-type distribution of particle size is obtained. Thus the present requirement that all pigment particles be less than one micron cannot be obtained for fluoride phosphors even when the average particle size is less than one micron without a separation process, which would be very difficult and expensive.
It is believed that small, uniform particle size up-converter fluoride phosphors would have superior luminescent efficiency over oxide or oxysulfide phosphors, and would be highly desirable for use in ink jet printer inks as well as for use in diagnostic assays.